Mutations in individual transcription factors (TFs) cause different disease states in particular cells and tissue types, suggesting that regulation of distinct target genes in a context-dependent way is critical for normal development and function. Despite decades of study of developmental gene regulation, it is still unclear how TFs expressed at different times and in different tissues regulate subsets of target genes in context-dependent ways. Furthermore, multiple TFs with similar binding preferences are often simultaneously expressed in the same cells, but how their individual activities are integrated is poorly understood. We propose here a detailed study of context-specific gene regulation by K50HD TFs, a functionally related group that share a critical Lysine (K) at position 50 of their homeodomains (K50HD) and bind a consensus TAATCC core. We will use the well-studied Drosophila embryo and eye as paradigms to address how each K50HD TF binds preferred alternative sites to control context-specific expression of target genes (Aim 1). The K50HD TFs Orthodenticle, Bicoid, and Goosecoid play roles in embryonic patterning, whereas Orthodenticle, Ptx1, Defective Proventriculus, and Goosecoid are important for eye development. We will use Protein-Binding Microarrays to rigorously define intrinsic DNA-binding preferences for each K50HD TF, and competitive gel shifts and transfection assays to verify preferred alternative binding sites. We will perform ChIP-exo (a modified version of ChIP-seq with higher resolution) to assess enrichment of preferred alternative sites in bound DNA regions. We will evaluate transcriptomes in wild type and mutant backgrounds to determine the impact of K50HD TF binding at alternative sites to gene regulation. We will then use enhancer reporter constructs to test the necessity and sufficiency of preferred alternative sites in context-specific regulation.In Aim 2, we will address how K50HD TFs work combinatorial with other factors to dictate context-specific expression. We will combine DNA binding and transcriptome data to define and characterize a large set of K50HD-dependent regulatory elements (enhancers) that drive expression in the embryo and/or the eye. After grouping enhancers according their differential expression patterns, we will search for over-represented motifs that dictate context-dependent expression. These motifs will be manipulated in transgenic assays to test regulatory functions, and used to identify trans-acting factors that control enhancer specificity via combinatorial mechanisms. Our findings will define how enhancers integrate the binding activities of multiple transcription factors to correctly activate batteries of target genes in tim and space. Our studies will also provide insights into how the fine details of enhancer architecture are critical for regulation by complex milieus of TFs to produce precise context-specific expression.